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WO2003018833A2 - 3-hydroxymethylglutaryl-coenzyme-a reductase et diagnostic et pronostic de la demence - Google Patents

3-hydroxymethylglutaryl-coenzyme-a reductase et diagnostic et pronostic de la demence Download PDF

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WO2003018833A2
WO2003018833A2 PCT/CA2002/001333 CA0201333W WO03018833A2 WO 2003018833 A2 WO2003018833 A2 WO 2003018833A2 CA 0201333 W CA0201333 W CA 0201333W WO 03018833 A2 WO03018833 A2 WO 03018833A2
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hmgr
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dementia
sample
gene
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WO2003018833A3 (fr
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Judes Poirier
Louise Theroux-Lamarre
Doris Dea
Nicole Aumont
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Mcgill University
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Priority to AU2002325705A priority patent/AU2002325705A1/en
Priority to US10/487,747 priority patent/US20050069881A1/en
Publication of WO2003018833A2 publication Critical patent/WO2003018833A2/fr
Publication of WO2003018833A3 publication Critical patent/WO2003018833A3/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to methods for the diagnosis and prognostication of dementia based on 3-hydroxymethylglutaryl coenzyme A reductase (HMGR) activity and expression.
  • HMGR 3-hydroxymethylglutaryl coenzyme A reductase
  • AD Alzheimer disease
  • AD is a progressive neurodegenerative disorder with clinical characteristics and pathological features.
  • AD is etiologically heterogenous and can be produced by mutations on genes localized on chromosomes 1, 14 and 21.
  • Major risk factors have been identified for the common form of AD (also refer to as sporadic AD) . These include the apolipoprotein E4 allele (chromosome 19) , butirylcholinesterase K
  • chromosome 3 alpha 2 -macroglobulin (chromosome 12), lipoprotein lipase (Baum et al . , 1999) and two of the apoE receptors called LRP (Beffert et al . , 1999a [chromosome 12]) and VLDL receptor (O uizumi et al . , 1995).
  • LRP Beffert et al . , 1999a [chromosome 12]
  • VLDL receptor O uizumi et al . , 1995.
  • the apolipoprotein E4 polymorphism was also shown to affect age of onset, rate of progression, cholinergic function and therapeutic response in AD.
  • Several other genetic risk factors have been identified in sporadic AD but replication has proven difficult for those novel risk markers. To date, there is no known genetic mutation responsible for the common form of AD.
  • AD Alzheimer disease
  • sporadic form of AD The familial form of AD accounts for roughly 10-15% of all cases worldwide, whereas the sporadic form of AD represents 85-90% of the remaining cases and is generally believed to be of late onset, occurring after 65 years of age.
  • AD amyloid precursor protein gene
  • presenilin genes mutations in the amyloid precursor protein gene and two presenilin genes
  • apolipoprotein E4 on chromosome 19 has been linked to both the late onset familial form, as well as to the sporadic form of AD.
  • the majority of patients referred to as sporadic cases probably arise as the result of several genetic anomalies, each making an independent contribution to the overall phenotype and pathophysiological process. It is suspected that at least one, and most probably several, additional mutations remain to be identified since only 50% of all AD cases have been linked to specific genetic anomalies in case/control studies.
  • the Amyloid Precursor Protein The first gene ever identified in association with familial AD was the amyloid precursor protein (APP) (Chartier-Harlin et al . , 1991).
  • the APP gene encodes a transcript which, once translated, encodes a single trans-membrane spanning polypeptide of roughly 750 amino acids. Alternative splicing of exon 7 and exon 8 results in a polypeptide of 695 amino acids, which is expressed at very high concentration in the central nervous system. APP is known to undergo a series of proteolytic cleavages which result in the production of a small 40 to
  • a beta peptide 42-amino acid long peptide referred to as the A beta peptide (Sisodia et al . , 1990).
  • the exact function of the amyloid precursor protein is currently unknown. Onset at around the age of 50 years is characteristic of familial AD pedigrees associated with mutation in the amyloid precursor protein gene: several mutations (including those at positions 665, 670, 673, 692, 693, 713, 716, 717) have been identified as mutations causing early to late onset familial AD. It has been proposed that these mutations in the APP gene cause an overproduction of the so-called neurotoxic form of beta amyloid referred as the 1-42 / 1-43 beta peptides. Polymerization of these fibres could then result in the development of senile plaque in the brain of AD patients with a concomitant impact on the brain integrity.
  • Presenilin 1 Following the discovery that only a portion of the familial cases of AD could be explained by the presence of a mutation in the amyloid precursor protein gene, several independent investigators pursued the hunt for other candidate genes that might be involved in the remaining familial forms of the disease.
  • the presenilin 1 gene localised on chromosome 14 (St George-Hyslop et al . , 1992) was then isolated using positional cloning strategy and more than 35 different mutations were identified by several independent groups as anomalies causing the familia-1 form of the disease.
  • the presenilin 1 gene is transcribed in several organs and in several cell types.
  • Presenilin 2 Following the cloning of the presenilin 1 gene on chromosome 14, a very similar sequence has been identified and subsequently localized on chromosome 1 (Levy- Lahad et al . , 1995). This polypeptide, referred to as presenilin 2, has an open reading frame of about 448 amino acids with substantial amino acid sequence identity with the presenilin 1 protein. Presenilin 2 appears to be more ubiquitously expressed but less abundant than presenilin 1. It has been proposed that presenilins may be involved in the intracellular trafficking and/or transport of specific proteins inside the cells. Mutational analysis in familial cases of AD uncovered two different missense mutations in the presenilin 2 gene in families segregating for early onset AD.
  • AD apolipoprotein E
  • the E4 allele of apoE although neither necessary nor sufficient to cause AD, is strongly associated with increased risk, rate of progression and severity of the neuropathology.
  • the effect of apoE4 appears additive such that heterozygotes and homozygotes are, three and eight times more likely, respectively, to be affected than controls.
  • the variation at the apoE locus accounts for at most 50% of the genetic variation in liability (Pericak-Vance and Haines, 1995) to develop the disorder and there must be other genetic variants that account for remaining risk.
  • Apolipoprotein E and Cholesterol Homeostasis in Alzheimer Disease are protein components of lipoprotein particles. The latter are macromolecular complexes that carry lipids such as cholesterol and phospholipids from one cell to another within a tissue or between organs. Some apolipoproteins regulate extracellular enzymatic reactions related to lipid homeostasis while others are ligands for cell surface receptors that mediate lipoprotein uptake into cells and their subsequent metabolism. ApoE is a component of several classes of plasma and cerebrospinal fluid lipoproteins. ApoE was shown to be synthesized and secreted by glial cells, predominantly astrocytes.
  • LDL low-density lipoprotein
  • VLDL very low-density receptor
  • LRP LDL receptor-related protein
  • apoE lipid homeostasis in the brain is underscored by the fact that major plasma apolipoproteins such as apoB and apoAI are not synthesized in the central nervous system.
  • FIG. 1 illustrates the role of apoE and its major receptors in the transport and recycling of cholesterol from dead or dying neurons to intact neurons undergoing synaptic remodeling and compensatory terminal outgrowth. It was shown that following neuronal cell loss and terminal differentiation in the CNS, large amounts of lipids are released from degenerating axon membranes and myelin (Fig. 1, #1) . In response, astrocytes (Fig.
  • HMGR 3, 3-hydroxy-methylglutaryl- CoA reductase
  • eukaryote cells obtain their cholesterol from two distinct sources: a) it is synthesized directly from acetyl-CoA through the so-called HMGR pathway or, b) it is imported through the apoE/apoB (LDL) receptor family via lipoprotein- complex internalization (for a review see Beffert et al . , 1998b) .
  • HMGR pathway acetyl-CoA
  • LDL lipoprotein- complex internalization
  • Much of the free cholesterol generated during synapse degradation is stored in astrocytes in the CNS and, in macrophages in the PNS where it is eventually reused during PNS regeneration and CNS reinnervation.
  • the apoE/lipoprotein complexes Following binding of the apoE/lipoprotein complexes with neuronal LDL receptors, the apoE/Lipoprotein/LDL receptor complex is internalized, degraded and the cholesterol released inside neurons where it is used for membrane synthesis and synaptic remodelling (Fig. 1, #3 and #4) .
  • the intracellular rise in cholesterol causes a down-regulation of HMG-CoA reductase activity and mRNA prevalence in granule cell neurons undergoing dendritic and synaptic remodeling (Fig. 1, #7) .
  • Apolipoprotein E, Cholesterol levels and the Amyloid Hypothesis of AD While the abnormal processing of the APP into toxic forms of beta amyloid appears to underlie the pathophysiological process that characterizes chromosome 1, 14 and 21 familial cases, the role of apoE as a potent scavenger of beta amyloid in the brain is certainly consistent with this working hypothesis (Beffert and Poirier, 1998; Beffert et al . , 1998b; Beffert et al . , 1999b) . For a while, it was generally believed that mutations in the apoE gene on chromosome 19 and in the APP gene on chromosome 21 represented independent biochemical pathways with similar outcomes; i.e. dementia of the Alzheimer type.
  • ApoE4 allele dosage was shown to modulate the age of onset of AD in families with the amyloid precursor protein (APP) mutation (Farrer et al . , 1997; Hardy, 1994).
  • apo E4 allele dose was shown to positively correlate with the density of beta A4 immunopositive plaques and neurofibrillary tangles in the cortex and hippocampus of AD subjects. Howland et al .
  • Mammalian cells particularly astrocytes and neurons, cultured in vi tro synthesize cholesterol at a rate which is inversely proportional to the cholesterol content in the growth medium.
  • Cholesterol requirements of most mammalian cells are met by two separate but interrelated processes.
  • One process is the endogenous synthesis of cholesterol. This synthesis pathway which involves over 20 reactions is regulated primarily by the activity of the 3-Hydroxy-3- MethylGlutaryl Coenzyme A Reductase (HMGR) which catalyzes the formation of mevalonate, the key precursor molecule in the synthesis of cholesterol.
  • HMGR 3-Hydroxy-3- MethylGlutaryl Coenzyme A Reductase
  • the other process involves the utilization of lipoprotein-derived cholesterol following internalization of the lipoprotein bound to its surface receptor (usually, an apoE-rich lipoprotein complex) .
  • Cholesterol homeostasis in brain cells is controlled by the perfect balance between cholesterol influx through the apoE receptors pathway and synthesis via the HMGR pathway, the rate limiting step in cholesterol biosynthesis.
  • the brain differs significantly from peripheral organs where a multitude of apolipoproteins such as apoB, apoH, apoAl, A2, apoCI and apoCII are playing a pro-active role in lipid transport and homeostasis.
  • the brain is entirely devoid of apoB (apoE's main back-up system in the blood) and contains only trace amounts of the other apos described above. For some unknown reason, the brain is extremely dependent on apoE and its accessory proteins to deliver and/or produce cholesterol in the intact or injured brain cells.
  • HMGR cholesterol synthesis via the HMGR pathway is required only when lipoprotein internalization by apoE/apoE receptor pathway is insufficient to meet the cholesterol requirement of the cell (Brown et al . , 1973; Rodwell et al . , 2000).
  • the endoplasmic reticulum-bound HMGR is regarded as the rate limiting step in the synthesis of cholesterol, a critical membrane lipid, precursor of steroid hormones (glucocorticoids and estrogen) and a signaling molecule involved in embryogenesis (Ness and Chambers, 2000) .
  • HMGR HMGR localized in the peroxisomal compartment
  • HMGR inhibitors such as simvastatin
  • the HMGR activity is down regulated in favor of uptake via apoE receptors (Sato and Takano, 1995) .
  • a similar process was reported in the PNS and CNS during the acute phase of regeneration that ensue degradation of dead cells following experimental injury (Boyles et al . , 1990) (Poirier et al . , 1993b; Poirier, 1994) .
  • HMGR protein The factor that has been shown to regulate the expression of the reductase is the controlled degradation of the HMGR protein (Gardner and Hampton, 1999; Cronin et al . , 2000). Lastly, there is evidence from hamster for a modulation in translation efficiency of mRNA for HMGR resulting in decreased or increased reductase protein and activity (Choi and Choi, 2000) .
  • the invention provides methods and commercial packages for the diagnosis and prognostication of a dementia based on an HMGR gene, its transcripts, and activity of HMGR protein.
  • a dementia is an Alzheimer disease.
  • the invention provides a method of diagnosing or prognosticating a dementia in a subject, said method comprising: (a) obtaining a sample from said subject, wherein said sample comprises nucleic acid comprising a 3- hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene; and (b) determining whether said nucleic acid comprises a polymorphism relative to a corresponding control sample obtained from a control subject; wherein the presence of said polymorphism is used to diagnose or prognosticate a dementia.
  • HMGR 3- hydroxy-3-methylglutaryl coenzyme A reductase
  • the above-mentioned polymorphism is localized in intron B (also known as intron 2, eg in hamsters) of said HMGR gene.
  • the above-mentioned step (b) comprises: (i) amplifying a nucleic acid sequence comprising said intron B (also known as intron 2, eg in hamsters) by polymerase chain reaction (PCR) to obtain a PCR product; (ii) digesting said PCR product with a restriction enzyme to obtain a restriction digest product; and (iii) determining a size of said restriction digest product.
  • the above-mentioned restriction enzyme is ScrFI .
  • the invention further provides a method of diagnosing or prognosticating a dementia in a subject, said method comprising: (a) measuring a first level of HMGR activity in a sample obtained from said subject; and (b) comparing said first level to a second level which is an average HMGR activity measured in at least one corresponding control sample obtained from at least one control subject, whereby if said first level is significantly less than said second level then said subject suffers from a dementia; wherein said method is used to diagnose or prognosticate a dementia.
  • the invention further provides a method of diagnosing or prognosticating a dementia in a subject, said method comprising: (a) obtaining a sample from said subject, wherein said sample comprises ribonucleic acid encoded by an HMGR gene; and (b) determining whether said sample comprises at least one alteration relative to a corresponding control sample obtained from a control subject, wherein said alteration is selected from the group consisting of: (i) an increase in a level of a first ribonucleic acid encoded by an HMGR gene, wherein said first ribonucleic acid has a deletion of exon 13; (ii) an increase in a level of a second ribonucleic encoded by an HMGR gene, wherein said second ribonucleic acid has an insertion of intron M; and (iii) a decrease in a level of a third ribonucleic acid comprising a normal HMGR transcript; wherein the presence of said at least one alteration is used to diagnose or
  • the above-mentioned alteration is determined using reverse transcriptase-polymerase chain reaction (RT-PCR) .
  • the invention further provides a commercial package for the diagnosis and/or the prognostication of a dementia, said commercial package comprising at least one of: (a) means for detecting a polymorphism in an HMGR gene in a sample together with instructions for assessing said polymorphism relative to a corresponding control sample; (b) means for determining a level of HMGR activity in a sample together with instructions for comparing said level with an established standard or a control level measured in at least one corresponding control sample; and (c) means for determining the presence of at least one feature selected from the group consisting of (i) a first HMGR ribonucleic acid having a deletion of exon 13, (ii) a second HMGR ribonucleic acid having an insertion of intron M, (iii) a decrease in a level of a third ribonucleic acid comprising
  • the above-mentioned sample is a tissue or body fluid of said subject.
  • the above-mentioned tissue or body fluid is neural tissue or fluid.
  • the above-mentioned tissue or body fluid is selected from saliva, hair, blood, plasma, lymphocytes, cerebrospinal fluid, epithelia and fibroblasts.
  • control subject is a normal age-matched subject.
  • above-mentioned method is used to prognosticate a dementia and wherein the control sample was obtained from the subject at another time.
  • the above-mentioned dementia is an Alzheimer disease.
  • Figure 1 Schematic representation of the postulated cascade of events regulating cholesterol transport during CNS reinnervation.
  • FC free cholesterol
  • CE cholesterol esters
  • E apoE.
  • FIG. 4 Genomic structure of the HMGR gene and its mRNA in the brain. Primers that were used for RT-PCR experiments to delineate exon 13 transcripts are shown.
  • Figure 5 HMGR transcript analysis via electrophoretic analysis of PCR products corresponding to each of the three transcripts identified (normal transcript; exon 13 deletion; and intron M insertion) .
  • FIG. 6 HMGR mRNA prevalence in the frontal cortex in Alzheimer disease. Prevalence of the three major forms (normal transcript; exon 13 deletion; and intron M insertion) in autopsy-confirmed AD versus control subjects.
  • Figure 7 Correlational analysis of the prevalence of the abnormal HMGR gene transcripts in the brain and the levels of toxic beta amyloid 1-40 in AD and age-matched control subjects.
  • FIG. 8 DNA Mutation and Polymorphism in the HMGR Gene in Alzheimer Disease
  • Figure 9 Sequencing results obtained from one specific Alzheimer disease patient in the vicinity of intron L and exon 13
  • Figure 10 PCR amplification of sequence-specific cDNA derived from mRNA extracted from autopsy-confirmed AD and control subjects wherein the upper band is representative of the HMGR mRNA transcript containing intron M.
  • Figure 11 Western blot analysis using a specific monoclonal antibody that recognizes a portion of the trans- membrane domain of HMGR as well as the catalytic site of HMGR.
  • HMGR 3-hydroxy-3-methylglutaryl-coenzyme A reductase
  • b) displays a significant polymorphic association with sporadic AD (genetic variation localized in intron B (also known as intron 2, eg in hamsters) of the HMGR gene; p ⁇ 0.01)
  • c) exhibits the presence of elevated concentrations of two abnormally processed RNAs in the brain of AD subjects : one mRNA lacks exon 13 whereas the other mRNA contains intron M (the intron between exon 12 and 13) .
  • d demonstrates brain levels of the toxic beta amyloid peptides 1-40 and 1-42 that markedly (and statistically) correlated with the increased proportion of HMGR mRNA containing the abnormal intron M and lacking exon 13.
  • HMGR gene which acts as a key accessory protein to the apoE/apoE receptor pathway and plays an active role in modulating cholesterol metabolism and beta amyloid production in vi tro, is defective in sporadic AD subjects.
  • the loss of HMGR activity is consistent with the reduction of cholesterol synthesis and levels in the AD brain as reported by several independent studies (Gottfries et al . , 1996a; Gottfries et al . , 1996b; Svennerholm and Gottfries, 1994; Svennerholm et al . , 1994; and data presented below) and its indirect effects on amyloid precursor protein metabolism (Bodovitz and Klein, 1996; Mills and Reiner, 1999; Simons et al . , 1998).
  • Applicant has examined candidate genes which are directly involved in the metabolism and/or function of the most important risk factor identified so far, namely the apolipoprotein E4 allele.
  • Applicants' findings presented herein indicate that the brain expresses three distinct mRNAs for the HMGR in contrast to human liver that normally produces only one form of the enzyme.
  • the presence of two transcripts has been reported previously in embryonic hamster cell line called UT-2 (Aboushadi et al . , 2000).
  • the shorter form of the mature mRNA is derived from exon 13 skipping. In the hamster, this shorter form codes for the HMGR localized in peroxisomes.
  • Results obtained in applicants' laboratory using real time quantitative RT-PCR amplification indicate that a very similar processing is occurring in the human brain with a short and long versions of the enzyme which exhibits (or lacks) exon 13 (based on the hamster gene nomenclature) .
  • Applicants have confirmed the nature of the different brain transcripts by laser sequencing.
  • HMGR mRNA containing the intron position between exon 12 and 13 increase proportionally to the relative concentration of the abnormal HMGR mRNA containing the intron position between exon 12 and 13. It is well known from genetic studies in the cardiovascular field that full HMGR knockout mice die prematurely in utero , whereas administration of a selective HMGR inhibitor called simvastatin directly into the brain causes lethality at high doses, but loss of white matter at low doses (a characteristic of Alzheimer and vascular dementia) .
  • transcript #1 an increase in the level of the two abnormal mRNAs transcript # 2 and transcript #3 relative to levels the normal HMGR mRNA (designated "transcript #1" in Example 6 below; i.e. containing exon 13 and lacking intron M) ; and (e) a correlation of the brain levels of beta amyloid peptides 1-40 and 1-42 with the increase in the presence of the two abnormal mRNAs transcript # 2 and transcript #3.
  • aspects of the present invention are methods for the diagnosis and prognostication of a dementia, such as an Alzheimer disease, via assessing whether a subject exhibits one of the above-mentioned events.
  • the invention provides a method of diagnosing or prognosticating a dementia, such as an Alzheimer disease, in a subject, said method comprising:
  • HMGR 3-hydroxy-3- methylglutaryl coenzyme A reductase
  • nucleic acid comprises a polymorphism relative to a corresponding control sample obtained from a control subject
  • the above-mentioned polymorphism is localized in intron B (also known as intron 2, eg in hamsters) of said HMGR gene.
  • intron B also known as intron 2, eg in hamsters
  • the polymorphism may be determined by methods known in the art.
  • the polymorphism may be determined via restriction fragment length polymorphism analysis (RFLP) , for example by amplifying a nucleic acid sequence comprising HMGR intron B (also known as intron 2, eg in hamsters) by polymerase chain reaction (PCR) to obtain a PCR product, digesting the PCR product with a restriction enzyme to obtain a restriction digest product; and examining the length of the restriction digest product.
  • a suitable restriction enzyme is ScrFI .
  • the invention further provides a method of diagnosing or prognosticating a dementia, such as an Alzheimer disease, in a subject, the method comprising:
  • the method is used to diagnose or prognosticate a dementia.
  • HMGR activity may be assessed or measured using methods known in the art.
  • Various means may be utilized to enable useful assay conditions. Such means may include, but are not limited to suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for HMGR activity and stability, temperature control means, and detection means to enable the detection of an HMGR reaction product.
  • the detection means detects cholesterol.
  • An example of a suitable method for determining HMGR activity is described in Poirier et al, 1993b.
  • the invention further provides methods of diagnosing or prognosticating a dementia, such as an Alzheimer disease, via the detection of abnormal HMGR mRNA transcripts #2 (lacking exon 13) and/or #3 (including intron M) noted above, and/or via assessing the levels of abnormal HMGR mRNA transcripts #2 and/or #3 relative to the level of the normal HMGR transcript #1 noted above (having exon 13 and lacking intron M)
  • the invention provides a method of diagnosing or prognosticating a dementia, such as an Alzheimer disease, in a subject, said method comprising:
  • mRNA transcripts may be detected by various methods known in the art. An example is detection using reverse transcriptase-polymerase chain reaction
  • RT-PCR which in an embodiment, is performed in a quantitative manner.
  • Transcripts may for example also be detected by Northern analysis using an appropriate probe (s)
  • the applicants' diagnostic method depends upon a comparison, with control levels, of the level of HMGR enzyme activity in postulated AD subjects, or upon a comparison of the nucleic acids encoding HMGR with those encoding its polymorphisms.
  • the enzyme activity control levels should be established based on analysis of corresponding tissues to the tissue of the AD subject which is being analyzed for HMGR activity. The appropriate tissue for analysis will depend upon the polymorphism as further described elsewhere herein, and more particularly upon whether what is being analyzed is HMGR activity or HMGR encoding nucleic acids.
  • a measured HMGR activity in an appropriate tissue sample obtained from an AD subject which exhibits a statistically significant reduction over the corresponding average level of HMGR activity in corresponding tissue of controls is a clear indication of dementia, particularly AD.
  • the above-mentioned sample is a tissue or body fluid of said subject, in a further embodiment, a neural tissue or fluid.
  • tissue or body fluids include but are not limited to saliva, hair, blood, plasma, lymphocytes, cerebrospinal fluid, epithelia, neural cells and fibroblasts.
  • control subject is a normal age-matched subject.
  • above mentioned methods are used for prognostication and the control sample is obtained from the subject at another time, in an embodiment, at an earlier time.
  • the above mentioned diagnostic and prognostic methods may be utilized independently or in further embodiments in various combinations.
  • the diagnostic and prognostic methods which detect polymorphisms of HMGR may be used to identify subjects at risk of developing a dementia, particularly an Alzheimer disease, and thereby permit appropriate precautionary or preventative treatment to be undertaken. Examples of such treatments include administering to such "at risk" subjects HMGR inhibitors such as statins.
  • the invention further relates to commercial packages or kits for carrying out the therapeutic, prophylactic, diagnostic and screening methods noted above, comprising the appropriate above-mentioned reagents together with instructions for methods of diagnosis and/or prognostication of a dementia, such as an Alzheimer disease.
  • the invention further provides a commercial package for the diagnosis and/or the prognostication of a dementia, such as an Alzheimer disease, said commercial package comprising at least one of:
  • the commercial package may be used for the analysis of samples as discussed above.
  • Example 1 Cholesterol levels in the brain of Alzheimer disease : Significant reduction of both free cholesterol and cholesterol esters (stored cholesterol) in the temporal cortex of autopsy-confirmed AD subjects.
  • Example 3 HMGR mRNA Prevalence in Temporal Cortex of AD Subjects
  • Figure 3 illustrates results obtained from quantitative real time PCR analysis of total HMGR mRNA prevalence in the frontal cortex of 23 AD subjects and 17 age-matched control subjects. E4 allele carriers as well as non-E4 subjects exhibit similar (lower) levels of total HMGR mRNAs.
  • the frontal cortex is an area of marked neuronal cell damage in Alzheimer disease.
  • Example 4 Analysis of a Polymorphic Marker in the HMGR Gene in Sporadic Alzheimer Disease.
  • the assay consists of a PCR amplification of the intron B (also known as intron 2, eg in hamsters) area followed by a digestion with the restriction enzyme ScrFI .
  • the digestion leads to the formation of 2 bands (heterozygotes) , one of which is 120 bp long and the other of which is 165 bp long (Leitersdorf et al . , 1990).
  • Table 2 summarizes the frequency distribution results obtained in applicants' pilot study with 84 autopsy-confirmed control and 64 AD subjects. Groups were matched for gender, age and ethnicity. The association between allele L and sporadic AD was estimated by chi-square analysis and found to be statistically significant (SPSS statistical program) .
  • the odds ratio is 1.8 with a 95% confidence interval of 1.1 to 3.1.
  • the autopsy-confirmed elderly control subjects do not differ from the published population prevalence. These results clearly indicate the L allele is linked to sporadic AD in this cohort of autopsy-confirmed subjects.
  • Example 5 Abnormal Processing of the HMGR mRNA in Alzheimer Disease.
  • HMGR mRNA anomalies in UT2 cells which are mutant clones of Chinese Hamster Ovary cells have been reported (Reynolds et al . , 1985) . UT2 cells are completely deficient in 97 Kd endoplasmic reticulum HMGR protein involved in cholesterol synthesis.
  • the defect arises from a mutation in the splice site causing exon 11 skipping (an mRNA without exon 11) .
  • the peroxisome HMGR protein and its mRNA are apparently intact in this cell type.
  • the mutation causes aberrant splice messages that are easily identifiable by PCR.
  • HMGR HMGR between exon 11 and 15 in AD and control subjects using the published sequence of the cDNA available in GENEBANK (Genebank accession number: L00166 and AH001819) .
  • This particular region of the mRNA contains the catalytic site of the enzyme.
  • the hamster genomic map was used as a reference to establish a working map of the human genomic structure (Genebank L00166 and AH001819) .
  • Applicants thus considered that exon skipping, insertion or deletion in one of the HMGR transcripts could explain the partial loss of activity observed in the AD brain without affecting the normal sterol-mediated up regulation of gene expression.
  • the human brain expresses two major mRNAs for the HMGR: one without exon 13 and one with exon 13 ( Figure 4) .
  • both control and AD subjects express the shorter form of the HMGR (the peroxisomal form) and long version (ER) of the transcript as shown in Figure 3.
  • Applicants have developed specific DNA primers to amplify the cDNA that contains the exon 13 insert, and another set of primers that amplifies only the transcript that lacks exon 13 ( Figure 4 and 5) .
  • Applicants have used a modified version of the method described by Powell and Kroon for HMGR mRNA quantification (Powell and
  • Figure 5 illustrates the corresponding band profile obtained after RT-PCR and gel electrophoresis of the mRNA containing (or not) exon 13. Applicants have repeated these analyses in a small group of parkinsonians to examine the HMGR transcript profile in another neurodegenerative disease characterized by glial cell proliferation.
  • Results were identical to the age-matched normal control subjects. Applicants repeated the analysis with 13 additional AD, 23 control and 10 parkinsonian subjects. Only AD subjects exhibit the intense band corresponding to exon 13 + insert. Furthermore, applicants have examined the HMGR transcript profile in the human glioblastoma cell line.
  • HTB-14 (ATCC collection: apoE genotype 3/3) and found the usual short and a long bands, without the abnormal "exon 13
  • HMGR transcripts there are two types.
  • One transcript contains exon 2 to 20 as expected whereas a second transcript contains exons 2-13, intron M, exons 14 to 20.
  • the second transcript which contains the entire intron M (which is localized between exon 13 and 14) appears to be the result of an abnormal splicing of the HMGR pro-transcript.
  • Primers were designed to amplify by real time quantitative RT-PCR, pieces of cDNA that corresponded to Transcripts #1,- #2 and #3. Another pair of primers was designed to quantify the total mRNA prevalence of the HMGR in the brain, irrespective of the exon 13 modifications ( Figure 6A) . This pair of primers amplifies a portion of exon 14.
  • the RT-PCR fragments corresponding to Trancripts #1, #2 and #3 are of similar length, with similar CG composition so that the proportion of each transcript species is determined in relation to the total HMGR mRNA prevalence.
  • beta actin levels were estimated in each samples and used as an internal, non changing, standard.
  • PD Parkinson disease
  • the AD brain is characterized by an increased prevalence of HMGR transcript without exon 13 (exon skipping) in brain areas damaged by the disease process.
  • Example 7 Beta amyloid, Alzheimer disease pathology and HMGR mRNA processing.
  • beta amyloid peptide is known to polymerize in the brain of Alzheimer subjects and to cause the accumulation of the so-called toxic beta amyloid deposits. Because of the well known contribution of beta amyloid to the pathophysiology of Alzheimer disease, applicants examined the effect of the alteration in HMGR transcript prevalence in relation to the production of the two major forms of beta amyloid in the brain of AD subjects: the beta amyloid 1-40 and the beta amyloid 1-42.
  • Figure 7 illustrates the correlational analysis that was performed using applicants' AD and control patient populations. Results clearly indicate that the increased production (and levels) of beta amyloid in the brain of AD subjects is tightly associated with the increased prevalence of the abnormal transcripts #2 or #3 (p ⁇ 0.005). In contrast, there is no association between the "normal HMGR transcript” prevalence and the levels of beta amyloid peptides in the brain of applicants' subjects (not shown) . The marked dichotomy between the control subjects (circles) and Alzheimer disease patients (triangles) is also noted.
  • AD subjects expressing the highest incidence of abnormal transcripts # 2 and #3 also exhibit the highest levels of beta amyloid 1-40 in the brain.
  • Example 8 Experimental methods.
  • HMGR intron B also known as intron 2, eg in hamsters
  • Alzheimer disease Assessment of the prevalence of the different polymorphisms in intron B (also known as intron 2, eg in hamsters) of the HMGR gene was performed as described by Leitersdorf, et al . (1990) using PCR amplification followed by restriction enzyme digestion (with ScfRI restriction enzyme) .
  • the prevalence of each allele (D and L) was determined for 64 Alzheimer disease subjects and 84 age-matched control subjects with no known neurological diseases. Aged subjects exhibited prevalence of D and L alleles similar to population studies published by Portersdorf et al . (1990) .
  • the AD cohort showed a statistically significant alteration of the D and L allele prevalence when compared to control cohorts.
  • Primer 18AS TGACTCTGCAGAAGTGAAAGCCTGGC
  • Primer 6GAS GTCCTTGCAGATGGGATGAC
  • RT-PCR technology allows us to monitor simultaneously different isoforms of a given mRNA and, to examine structural instability of the different isoforms by means of temperature dissociation profile.
  • the dissociation profile allows the amplification of the normal and abnormal transcripts, and to independently assess the relative amount of each transcript in a given brain area.
  • actin was used as positive control transcripts.
  • Enzymatic activity for HMGR in the brain was assessed as described in Poirier et al . , 1993b.
  • Example 9 HMGR genetic variants.
  • Figure 8 illustrates several of the polymorphisms found in applicants' analysis of the HMGR DNA of various Alzheimer disease subjects.
  • the DNA of one of applicants' AD subjects (known as subject 993) is characterized by the presence of a mutation at the frontier of intron L and exon 13, in the so-called splice site junction sequence.
  • This mutation replaces a key guanine (G) by an adenosine (A) in the consensus splice site between intron L and exon 13.
  • G key guanine
  • A adenosine
  • This anomaly which is heterozygous, i.e. present in only one allele
  • This type of intron retention due to splice site mutations has been described for other diseases, but never before for Alzheimer disease.
  • this anomaly involves a DNA mutation, and thus can be identified by a DNA test, this anomaly is readily detectable, not only in the brain which is affected by Alzheimer disease, but also in other tissues which do not exhibit symptoms of the disease, such as saliva, blood and hair.
  • the other polymorphisms described in Figure 8 (as identified by laser sequencing) in the introns I, J, K are also examples of genetic modifications in the HMGR transcript.
  • Figure 9 is a printout of the sequencing results obtained from Alzheimer disease patient 993 in the vicinity of intron L and exon 13.
  • a mutation at position 2 of the splice site junction (A versus G) is known to cause intron retention (and some times also exon skipping) in several independent genes in humans and animals.
  • this subject is homozygous for allele L in intron B (also known as intron 2, eg in hamsters) , which is the allele applicants have found to be strongly associated to common Alzheimer disease. It is thus no coincidence that both genetic anomalies are strongly linked to AD since they affect HMGR processing in a manner consistent with the presence of abnormal protein and loss of HMGR activity.
  • Figure 9 illustrates the sequencing output of the DNA sequencer demonstrating the presence of the two alleles (A or G) in the DNA of subject 993 suffering from Alzheimer disease. This 'finding provides an explanation (at least for this particular patient) of why Alzheimer disease subjects demonstrate abnormal splicing of the HMGR gene, causing intron M retention in all AD subjects examined so far. Because the mutation was found at the level of the DNA, genetic tests designed to identify mutations like these in the HMGR gene could be performed in any type of human tissue including blood, biopsy or hair.
  • Applicants have expanded on their original observation of the presence of the abnormal intron M in the mRNA of the HMGR.
  • Figure 11 examines the consequence of the presence of the abnormal transcript on the HMGR protein profile by Western • blot analysis using a specific monoclonal antibody that recognizes a portion of the trans-membrane domain of the enzyme as well as the catalytic site of the enzyme. Since the activity of the enzyme was shown to be markedly reduced in the Alzheimer brains, applicant expected (and found) the presence of abnormal bands in the western blot analysis. A band can be observed on top of the expected HMGR doublet at 36Kd and in some patients, another abnormal band can be devisved below the 36 Kd reference HMGR bands.
  • the high molecular weight band corresponds to the approximate molecular weight of a truncated form of the HMGR protein that retains the intron M which exhibits a stop codon in the first few nucleic acids of the intron. That truncated form of the HMGR protein is not only expressed in areas of the brain where the disease is most severe but also in the cerebellum, an area relatively free of pathology. This observation of a widespread expression of a truncated form of the HMGR in the brain of AD brain is very consistent with a genetic (widespread) defect causing abnormal splicing of the HMGR transcript and expression of a defective protein. This mutation in the DNA can be observed in samples taken from tissues throughout the subject's body, for example in the brain, cerebellum, liver, hair, saliva, hair and blood.
  • this mutation in combination with the modifications in intron B (also known as intron 2, eg in hamsters) described earlier, are clear examples of mutation/genetic anomalies that are detected by genetic testing or screening of DNAs and linked to Alzheimer disease.
  • the abnormal splice site mutation discovered at the junction of intron L and exon 13 also represents an example of a mutation that causes intron retention which in turn affects mRNA splicing, causing abnormal protein production and finally, loss of enzymatic activity and function. Since exons 12, 13 and 14 are the regions which give rise to the catalytic site of the enzyme, other mutations/polymorphisms in these regions of the HMGR gene will be of particular utility in applicants' diagnostic method.
  • Plastic neuronal remodeling is impaired in patients with Alzheimer's disease carrying apolipoprotein epsilon 4 allele. JNS 17, 516-529.
  • Apolipoprotein E uptake is increased by beta-amyloid peptides and reduced by blockade of the LDL receptor.
  • Neurodegenerative Diseases 103-108. Beffert, U., Aumont,N., Dea,D., Lussier-Cacan, S. , Davignon,J., and Poirier, J. (1998b). Beta-amyloid peptides increase the binding and internalization of apolipoprotein E to hippocampal neurons. J. Neurochem. 70, 1458- 1466. Beffert, U. , Aumont,N., Dea,D., Lussier-Cacan, S . , Davignon,J., and Poirier, J. (1999b). Apolipoprotein E isoform-specific reduction of extracellular amyloid in neuronal cultures. Molecular Brain Research 68 , 181-185.
  • Apolipoprotein E and beta-amyloid levels in the hippocampus and frontal cortex of Alzheimer's disease subjects are disease-related and apolipoprotein E genotype dependant. Brain Research 843, 87-94.
  • Hyslop,P.H. (1998). Association between bleomycin hydrolase and Alzheimer's disease in Caucasians. Ann. Neurol. 44, 808- 811.
  • Frimpong,K. and Rodwell,V.W. (1994) The active site of hamster 3-hydroxy-3-methylglutaryl-CoA reductase resides at the subunit interface and incorporates catalytically essential acidic residues from separate polypeptides. J. Biol. Chem. 269, 1217-1221.
  • HMG-CoA 3-hydroxy-3- methylglutaryl- coenzyme A
  • T/G polymorphism at intron 9 of presenilin 1 gene is associated with, but not responsible for sporadic late-onset Alzheimer's disease in Japanese population. Neurosci. Lett. 227, 123-126.
  • VLDL very low density lipoprotein
  • HMG CoA reductase a negatively regulated gene with unusual promoter and 5' untranslated regions. Cell 38, 275-285.
  • Presenilin-1 intron 8 polymorphism is not associated with autopsy-confirmed late-onset Alzheimer's disease. NL 222, 68-69.
  • Apolipoprotein E binding to soluble proteins
  • Alzheimer's beta-amyloid Biochemical & Biophysical Research Communications 192, 359-365.

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Abstract

Méthodes et kit commercial pour le diagnostic et / ou le pronostic de la démence. Lesdites méthodes sont basées sur l'évaluation d'une ou plusieurs caractéristiques relatives au gène 3-hydroxyméthylglutaryl-coenzyme-A réductase (HMGR), d'un polymorphisme associé au gène, de la nature des produits de transcription ARNm produits et / ou de l'activité protéique de HMGR. Les déposants ont déterminé qu'une baisse de l'activité de HMGR correspond à la maladie d'Alzheimer. Ils ont en outre déterminé que la présence d'un polymorphisme dans le gène HMGR correspond à la maladie d'Alzheimer. Ils ont encore identifié deux produits de transcription ARNm anormaux de HMGR et montré que leur présence correspond à la maladie d'Alzheimer.
PCT/CA2002/001333 2001-08-29 2002-08-29 3-hydroxymethylglutaryl-coenzyme-a reductase et diagnostic et pronostic de la demence WO2003018833A2 (fr)

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WO2009138130A1 (fr) * 2008-05-16 2009-11-19 Atlas Antibodies Ab Pronostic du cancer du sein

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US8084209B2 (en) * 2005-07-22 2011-12-27 Children's Hospital & Research Center Oakland HMGCR isoforms in prediction of efficacy and identification of cholesterol-modulating compounds
WO2008019187A2 (fr) * 2006-05-30 2008-02-14 Mayo Foundation For Medical Education And Research Détection et traitement de la démence
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WO2009138130A1 (fr) * 2008-05-16 2009-11-19 Atlas Antibodies Ab Pronostic du cancer du sein
US8945832B2 (en) 2008-05-16 2015-02-03 Atlas Antibodies Ab Treatment prediction involving HMGCR

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